The dendritic cells (DCs) of the mammalian innate immune system are responsible for detection of microbial encounters and the initiation of inflammation and adaptive immunity. This central role of DCs in host physiology has attracted much attention, and these cells are the subject of active investigation. Despite the appreciation of the importance of DCs, there remain large and fundamental gaps in our knowledge of their mechanisms of regulation. For instance, DCs encode several receptors that detect bacterial lipopolysaccharides (LPS), yet the functions of only one (TLR4) have been examined extensively. Recent work by us and others have identified the LPS receptors CD14 and caspase-11 as being capable of inducing novel signaling pathways that proceed either upstream of TLR4 (CD14) or in parallel to TLR4 (caspase-11). The collective actions of CD14, TLR4 and caspase-11 are important for DC activation and host defense, yet their mechanisms of action are poorly defined. Thus, significant gaps in our knowledge exist to explain the earliest stages of DC interactions with bacteria. We also lack an understanding of how DCs interact with T cells at later stages of infection, yet it is clear that these interactions lead to profound changes in the activities of both cell types. Much of the research into these interactions has focused on how DCs promote changes in T cell activities. In contrast, we have a minimal understanding of how T cells influence the activity of DCs. In this application, we propose to fill these gaps in our knowledge of DC biology through forward genetic screening for novel regulatory factors in mice. This approach is facilitated by recent advances in genome editing provided by CRISPR-based technologies. A pipeline of gene discovery will be generated through the use of established and emerging FACS-based assays. These assays will be used in vivo and ex vivo to identify DCs that are deficient for regulators of DC interactions with microbes or T cells. All screens will depend on significant interactions between the informatics, mouse perturbation and CRISPR library Cores associated with this U19 application. Subsequent functional analysis of candidate regulatory proteins will be performed in collaboration with other investigators on this grant. The cumulative result of these efforts will be a series of novel gene sets that should define pathways and processes that explain numerous aspects of DC biology as they relate to host defense.